The structure of receptor-associated protein (RAP)

The receptor-associated protein (RAP) is a molecular chaperone that binds tightly to certain newly synthesized LDL receptor family members in the endoplasmic reticulum (ER) and facilitates their delivery to the Golgi. We have adopted a divide-and-conquer strategy to solve the structures of the individual domains of RAP using NMR spectroscopy. We present here the newly determined structure of domain 2. Based on this structure and the structures of domains 1 and 3, which were solved previously, we utilized experimental small-angle neutron scattering (SANS) data and a novel simulated annealing protocol to characterize the overall structure of RAP. The results reveal that RAP adopts a unique structural architecture consisting of three independent three-helix bundles that are connected by long and flexible linkers. The flexible linkers and the quasi-repetitive structural architecture may allow RAP to adopt various possible conformations when interacting with the LDL receptors, which are also made of repetitive substructure units.     

Identification of the minimal functional unit in the low density lipoprotein receptor-related protein for binding the receptor-associated protein (RAP). A conserved acidic residue in the complement-type repeats is important for recognition of RAP

The low density lipoprotein receptor-related protein (LRP), a member of the low density lipoprotein receptor family, mediates the internalization of a diverse set of ligands. The ligand binding sites are located in different regions of clusters consisting of approximately 40 residues, cysteine-rich complement-type repeats (CRs). The 39-40-kDa receptor-associated protein, a folding chaperone/escort protein required for efficient transport of functional LRP to the cell surface, is an antagonist of all identified ligands. To analyze the multisite inhibition by RAP in ligand binding of LRP, we have used an Escherichia coli expression system to produce fragments of the entire second ligand binding cluster of LRP (CR3-10). By ligand affinity chromatography and surface plasmon resonance analysis, we show that RAP binds to all two-repeat modules except CR910. CR10 differs from other repeats in cluster II by not containing a surface-exposed conserved acidic residue between Cys(IV) and Cys(V). By site-directed mutagenesis and ligand competition analysis, we provide evidence for a crucial importance of this conserved residue for RAP binding. We provide experimental evidence showing that two adjacent complement-type repeats, both containing a conserved acidic residue, represent a minimal unit required for efficient binding to RAP.     

Minimization of the third domain of the LDL receptor-associated protein (RAP)

The third domain of the low-density lipoprotein receptor-associated protein (RAP d3) binds with high-affinity to pairs of complement-type repeats (CR) within the LDLR family of receptors. Structural analyses have defined the contact surface between RAP d3 and a CR pair from the low-density lipoprotein receptor (LDLR). Much of the sequence of RAP d3 has been proposed to stabilize the receptor-binding region without participating directly in formation of the contact surface. We have developed a truncated version of RAP d3 in which these scaffolding regions are excised and replaced with a single, intramolecular disulfide bond. This substitution allows for deletion of as much as a third of the RAP d3 sequence with substantial retention of receptor-binding ability.     

Assembly of very low density lipoprotein in the hepatocyte. Differential transport of apoproteins through the secretory pathway

The transport of the apolipoprotein (apo) constituents of hepatic very low density lipoprotein (VLDL) through the secretory pathway was investigated with estrogen-induced chick hepatocytes in primary culture. Cell monolayers were pulse-labeled with [3H]leucine and, after differing periods of chase with unlabeled leucine, were subjected to subcellular fractionation for 3H-apoprotein analysis. The first-order rate constants for transit of apoB, apoA-I, and apoII through the endoplasmic reticulum (ER) and Golgi were estimated using a three-compartment (ER, Golgi, and extracellular medium) kinetic analysis. The results indicate that apoB resides in the ER (t1/2 = 26 min) for a shorter period of time than in the Golgi (t1/2 = 43 min). For apoII, the t1/2 for transport through the ER and Golgi are 43 and 49 min, respectively. ApoA-I transits the ER at a rate (t1/2 = 6 min) much faster than apoB, apoII, and virtually all other secretory proteins. Upon reaching the Golgi, the rate of movement of apoA-I is markedly reduced (t1/2 = 28 min). Thus, in contrast to current models of protein secretion, the rate-limiting step in the secretion of VLDL apoproteins from the cell is transport through the Golgi, not the ER. Examination of the steady-state distribution of the apoproteins in the ER and Golgi support this conclusion. To characterize the intracellular transport process further, the distribution of apoproteins between the lumenal contents of the ER and Golgi and the membranes which delineate these compartments was determined after steady-state labeling with [3H]leucine. Approximately 50% of the apoB in the ER and in a dense, early Golgi fraction was membrane-associated, whereas in a less dense or late Golgi compartment, only 20% was bound to membranes. ApoII was also associated with the membranes of the ER and Golgi to a significant extent. In contrast, apoA-I was primarily localized lumenally throughout the secretory pathway. The occurrence of membrane-associated apoproteins in the Golgi, coupled with their slow rate of transit through this compartment suggests a major role for the Golgi in the assembly of the constituents of VLDL, and suggests that interaction of apoproteins (apoB) with the membranes of the Golgi is required for the maturation of VLDL.     

High affinity binding of receptor-associated protein to heparin and low density lipoprotein receptor-related protein requires similar basic amino acid sequence motifs

The 39-kDa receptor-associated protein (RAP) is a specialized chaperone for members of the low density lipoprotein receptor gene family, which also binds heparin. Previous studies have identified a triplicate repeat sequence within RAP that appears to exhibit differential functions. Here we generated a series of truncated and site-directed RAP mutants in order to define the sites within RAP that are important for interacting with heparin and low density lipoprotein receptor-related protein (LRP). We found that high affinity binding of RAP to heparin is mediated by the carboxyl-terminal repeat of RAP, whereas both the carboxyl-terminal repeat and a combination of amino and central repeats exhibit high affinity binding to LRP. Several motifs were found to mediate the binding of RAP to heparin, and each contained a cluster of basic amino acids; among them, an intact R(282)VSR(285)SR(287)EK(289) motif is required for high affinity binding of RAP to heparin, whereas two other motifs, R(203)LR(205)R(206) and R(314)ISR(317)AR(319), also contribute to this interaction. We also found that intact motifs of both R(203)LR(205)R(206) and R(282)VSR(285)SR(287)EK(289) are required for high affinity binding of RAP to LRP, with the third motif, R(314)ISR(317)AR(319), contributing little to RAP-LRP interaction. We conclude that electrostatic interactions likely contribute significantly in the binding of RAP to both heparin and LRP and that high affinity interaction with both heparin and LRP appears to require mostly overlapping sequence motifs within RAP.     

Dominant thermodynamic role of the third independent receptor binding site in the receptor-associated protein RAP.

The 39 kDa receptor-associated protein (RAP) is a three-domain escort protein in the secretory pathway for several members of the low-density lipoprotein receptor (LDLR) family of endocytic receptors, including the LDLR-related protein (LRP). The minimal functional unit of LRP required for efficient binding to RAP is composed of complement-type repeat (CR)-domain pairs, located in clusters on the extracellular part of LRP. Here we investigate the binding of full-length RAP and isolated RAP domains 1-3 to an ubiquitin-fused CR-domain pair consisting of the fifth and sixth CR domains of LRP (U-CR56). As shown by isothermal titration calorimetric analysis of simple RAP domains as well as adjoined RAP domains, all three RAP domains bind to this CR-domain pair in a noncooperative way. The binding of U-CR56 to RAP domains 1 and 2 is (at room temperature) enthalpically driven with an entropy penalty (K(D) = 2.77 x 10(-6) M and 1.85 x 10(-5) M, respectively), whereas RAP domain 3 binds with a substantially lower enthalpy, but is favored due to a positive entropic contribution (K(D) = 1.71 x 10(-7) M). The heat capacity change for complex formation between RAP domain 1 and the CR-domain pair is -1.65 kJ K(-1) mol(-1). There is an indication of a conformational change in RAP domain 3 upon binding in the surface plasmon resonance analysis of the interaction. The different mechanisms of binding to RAP domains 1 and 3 are further substantiated by the different effects on binding of mutations of the Asp and Trp residues in the LRP CR5 or CR6 domains, which are important for the recognition of several ligands.     

Allosteric modulation of ligand binding to low density lipoprotein receptor-related protein by the receptor-associated protein requires critical lysine residues within its carboxyl-terminal domain

The low density lipoprotein receptor-related protein (LRP) is a large endocytic receptor that recognizes more than 30 different ligands and plays important roles in protease and lipoprotein catabolism. Ligand binding to newly synthesized LRP is modulated by the receptor-associated protein (RAP), an endoplasmic reticulum-resident protein that functions as a molecular chaperone and prevents ligands from associating with LRP via an allosteric-type mechanism. RAP is a multidomain protein that contains two independent LRP binding sites, one located at the amino-terminal portion of the molecule and the other at the carboxyl-terminal portion of the molecule. The objective of the present investigation was to gain insight into how these two regions of RAP interact with LRP and function to modulate its ligand binding properties. These objectives were accomplished by random mutagenesis of RAP, which identified two critical lysine residues, Lys-256 and Lys-270, within the carboxyl-terminal domain that are necessary for binding of this region of RAP to LRP and to heparin. RAP molecules in which either of these two lysine residues was mutated still bound LRP but with reduced affinity. Furthermore, the mutant RAPs were significantly impaired in their ability to inhibit alpha(2)M* binding to LRP via allosteric mechanisms. In contrast, the mutant RAP molecules were still effective at inhibiting uPA.PAI-1 binding to LRP. These results confirm that both LRP binding sites within RAP cooperate to inhibit ligand binding via an allosteric mechanism.     

Binding of low density lipoproteins (LDL) to platelets

Binding of LDL to platelets was studied by two independent methods, radioactive and flow cytofluorimetry, using 125I- and fluorescently labelled RITC-LDL. Saturation of 125I- and RITC-LDL binding to platelets, inhibition of binding by unlabelled LDL and a lower inhibitory effect of unlabelled HDL evidence the existence of a limited number of binding sites specific for LDL in platelets. Unlike nuclear cells platelets do not degrade LDL. The binding of LDL to platelets is reversible and independent of Ca2+. The decrease of total binding level at 4 degrees and the absence of heparin effect on the release of bound LDL suggest LDL incorporation into platelets.     

Thymus fraction (FIII) effect on cholesterol metabolism: modulation of the low density lipoprotein receptor pathway

1. A heat stable protein fraction from calf thymus is able to determine a marked drop of total serum cholesterol and B apoproteins in rats fed a cholesterol enriched diet. 2. It is known that the catabolism of low density lipoprotein (LDL) cholesterol is mediated by the existence of high affinity surface receptor on membrane cells. 3. The purpose of the present paper is to verify if the heat stable thymus protein fraction is able to affect the LDL receptor pathway in vitro. 4. The results obtained show that the protein calf thymus is able to increase human and rat LDL catabolism.     

LRP6 protein regulates low density lipoprotein (LDL) receptor-mediated LDL uptake

Genetic variations in LRP6 gene are associated with high serum LDL cholesterol levels. We have previously shown that LDL clearance in peripheral B-lymphocytes of the LRP6(R611C) mutation carriers is significantly impaired. In this study we have examined the role of wild type LRP6 (LRP6(WT)) and LRP6(R611C) in LDL receptor (LDLR)-mediated LDL uptake. LDL binding and uptake were increased when LRP6(WT) was overexpressed and modestly reduced when it was knocked down in LDLR-deficient CHO (ldlA7) cells. These findings implicated LRP6 in LDLR-independent cellular LDL binding and uptake. However, LRP6 knockdown in wild type CHO cells resulted in a much greater decline in LDL binding and uptake compared with CHO-ldlA7 cells, suggesting impaired function of the LDLR. LDLR internalization was severely diminished when LRP6 was knocked down and was restored after LRP6 was reintroduced. Further analysis revealed that LRP6(WT) forms a complex with LDLR, clathrin, and ARH and undergoes a clathrin-mediated internalization after stimulation with LDL. LDLR and LRP6 internalizations as well as LDL uptake were all impaired in CHO-k1 cells expressing LRP6(R611C). These studies identify LRP6 as a critical modulator of receptor-mediated LDL endocytosis and introduce a mechanism by which variation in LRP6 may contribute to high serum LDL levels.     

Essential role of the low density lipoprotein receptor-related protein in vascular smooth muscle cell migration

The low density lipoprotein receptor-related protein (LRP) is a multifunctional cell surface receptor highly expressed in human aortic smooth muscle cells. In the present study, we used the short interfering RNA (siRNA) technique to explore the role of LRP in smooth muscle cell migration. We identified an LRP-specific siRNA that selective silences LRP expression in human aortic smooth muscle cells. As a consequence, LRP-mediated ligand degradation was significantly reduced. More important, we found that platelet-derived growth factor-dependent cell migration was inhibited in cells transfected with LRP siRNA. These results demonstrate an important role of LRP in smooth muscle cell migration.     

A protective role of the low density lipoprotein receptor-related protein against amyloid beta-protein toxicity

In order to delineate the neuroprotective role of the low density lipoprotein receptor-related protein (LRP) against amyloid beta-protein toxicity, studies were performed in C6 cells challenged with amyloid beta-protein in the presence or absence of activated alpha(2)-macroglobulin. Toxicity was assessed via two cell viability assays. We found that this endocytic receptor conferred protection against amyloid beta-protein toxicity in the presence of activated alpha(2)-macroglobulin and its down-regulation via inhibition by receptor-associated protein or transfection of cells with presenilin 1, increased susceptibility to amyloid beta-protein toxicity. Increased surface LRP immunoreactivity in response to amyloid beta-protein challenge was associated with increased translocation of LRP from the endoplasmic reticulum to the surface, rather than from increased mRNA or protein expression. Furthermore, this translocation of LRP to the surface was mediated by a calcium/calmodulin protein kinase II-dependent signaling pathway. These studies provide evidence for a protective role of LRP against amyloid beta-protein toxicity and may explain the aggressive nature of presenilin-1 mutation in familial Alzheimer's disease.     

Binding of thyroxine to human plasma low density lipoprotein through specific interaction with apolipoprotein B (apoB-100)

Human plasma low density lipoprotein (LDL), which binds 0.2% of plasma T4, was shown to interact with the hormone through its protein moiety, apolipoprotein B-100. LDL and LDL2, the major subfraction of LDL, were found to have 3 equivalent binding sites for T4 with Ka = 2.5 x 10(6) M-1. Photoaffinity labeling of LDL with inner ring-labeled [125I]T4, followed by SDS-PAGE or agarose-SDS-PAGE of the labeled products, revealed that apoB-100 and its proteolytic cleavage products, apoB-74 and apoB-26, bound [125I]T4. In the presence of 1 or 10 microM T4, labeling was decreased in 7 separate experiments by 40-53% or 65-86%, respectively, consistent with a Ka of approximately 10(6) M-1. Binding of T4 to apoB-100 associated with VLDL was also demonstrated by photoaffinity labeling. The observed thyroid hormone binding property of lipid-complexed apoB-100 and the knowledge that receptors for the apolipoprotein exist in various tissues suggest a possible physiological role in thyroid hormone transport.     

Internalization of transthyretin. Evidence of a novel yet unidentified receptor-associated protein (RAP)-sensitive receptor

Transthyretin (TTR) is a plasma carrier of thyroxine and retinol-binding protein (RBP). Though the liver is the major site of TTR degradation, its cellular uptake is poorly understood. We explored TTR uptake using hepatomas and primary hepatocytes and showed internalization by a specific receptor. RBP complexed with TTR led to a 70% decrease of TTR internalization, whereas TTR bound to thyroxine led to a 20% increase. Different TTR mutants showed differences in uptake, suggesting receptor recognition dependent on the structure of TTR. Cross-linking studies using hepatomas and (125)I-TTR revealed a approximately 90-kDa complex corresponding to (125)I-TTR bound to its receptor. Given previous evidence that a fraction of TTR is associated with high-density lipoproteins (HDL) and that in the kidney, megalin, a member of the low-density lipoprotein receptor family (LDLr) internalizes TTR, we hypothesized that TTR and lipoproteins could share related degradation pathways. Using lipid-deficient serum in uptake assays, no significant changes were observed showing that TTR uptake is not lipoprotein-dependent or due to TTR-lipoprotein complexes. However, competition studies showed that lipoproteins inhibit TTR internalization. The scavenger receptor SR-BI, a HDL receptor, and known LDLr family hepatic receptors did not mediate TTR uptake as assessed using different cellular systems. Interestingly, the receptor-associated protein (RAP), a ligand for all members of the LDLr, was able to inhibit TTR internalization. Moreover, the approximately 90-kDa TTR-receptor complex obtained by cross-linking was sensitive to the presence of RAP. To confirm that RAP sensitivity observed in hepatomas did not represent a mechanism absent in normal cells, primary hepatocytes were tested, and similar results were obtained. The RAP-sensitive TTR internalization together with displacement of TTR uptake by lipoproteins, further suggests that a common pathway might exist between TTR and lipoprotein metabolism and that an as yet unidentified RAP-sensitive receptor mediates TTR uptake.     

The receptor-associated protein (RAP) interacts with several resident proteins of the endoplasmic reticulum including a glycoprotein related to actin

The receptor-associated protein (RAP) is a chaperone found primarily in the endoplasmic reticulum (ER) that plays a necessary role in the folding and exocytic trafficking of members of the LDL receptor gene family including megalin and the LDL receptor-related protein (LRP). Recently, RAP has been shown to interact with a growing number of proteins including several that are unrelated to the LDL receptor family as well as new members of this rapidly expanding family. Based on these observations, we have applied chemical crosslinking procedures to identify additional novel RAP-interacting proteins, and thereby better characterize the scope of RAP's ER-related function. In this study, we have identified eight proteins with molecular weights of 32, 35, 46, 55, 70, 95, 170, and 200 kDa that interact with endogenous RAP. These proteins were found to associate with RAP in multiple cell types from different species, suggesting that their expression and interactions with RAP are ubiquitous. Results of pulse-chase experiments show that most of the proteins remain sensitive to endoglycosidase-H digestion, and also remain stably associated with RAP over an extended period, suggesting that they are ER resident proteins. All of the RAP-associated proteins appear to be largely soluble as they partition into the aqueous phase following TX-114 detergent extraction. Sequence analysis and immunoblotting of the 46-kDa RAP-associated glycoprotein (gp46) shows that it is structurally and immunologically related to actin. If gp46 is also functionally related to actin as an intracellular structural protein, it may represent a novel component of the putative ER matrix.     

Distinct properties of the recognition sites for beta-very low density lipoprotein (remnant receptor) and alpha 2-macroglobulin (low density lipoprotein receptor-related protein) on rat parenchymal cells.

The properties of the recognition sites for alpha 2-macroglobulin (alpha 2-macroglobulin receptor; low density lipoprotein receptor-related protein) and beta-migrating very low density lipoprotein (beta-VLDL) (remnant receptor) on rat parenchymal cells were directly compared to analyze whether both substrates are recognized and internalized by the same receptor system. In cholesterol-fed rats, the large circulating pool of beta-VLDL is unable to diminish the liver uptake of 125I-labeled alpha 2-macroglobulin, while liver uptake of 125I-labeled beta-VLDL in these rats is reduced by 87.3% at 10 min after injection. In vitro competition studies with isolated parenchymal liver cells demonstrate that the binding of 125I-labeled alpha 2-macroglobulin to rat parenchymal cells is not effectively competed for by beta-VLDL, whether this lipoprotein is additionally enriched in apolipoprotein E or not. Binding of alpha 2-macroglobulin to parenchymal cells requires the presence of calcium, while binding of beta-VLDL does not. Incubation of parenchymal cells for 1 h with proteinase K reduced the subsequent binding of alpha 2-macroglobulin by 90.1%, while the binding of beta-VLDL was reduced by only 20.2%. In the presence of monensin, the association of alpha 2-macroglobulin to parenchymal cells at 2 h of incubation was reduced by 64.7%, while the association of beta-VLDL was not affected. Preincubation of parenchymal cells with monensin for 60 min at 37 degrees C reduced the subsequent binding of alpha 2-macroglobulin by 54.5%, while binding of beta-VLDL was only reduced by 14.6%. The results indicate that the recognition sites for alpha 2-macroglobulin and beta-VLDL on rat parenchymal cells do exert different properties and are therefore likely to reside on different molecules.